The importance of the Notch-Atoh1 molecular pathway in colorectal cancer (CRC) tumorigenesis has recently being recognized, suggesting that this pathway is a target for new CRC therapeutics. Our preliminary data suggest that ATOH1 is a critical gatekeeper for the program of Notch-directed differentiation of intestinal stem cells, and that ATOH1 functions as a tumor suppressor in CRC where it is silenced by methylation and deletion. Furthermore, our data show that Notch inhibitory drugs-which force differentiation and block proliferation in colon cells-require ATOH1 for these effects. We also identified SPDEF as a downstream target of ATOH1 and showed that SPDEF inhibits intestinal epithelial proliferation. Taken together, these results suggest that ATOH1 may be the key target of the Notch pathway regulating differentiation and proliferation in CRCs via SPDEF. This work will test the hypothesis that ATOH1 is a colon tumor suppressor that mediates anti- cancer effects of Notch inhibitors via SPDEF. In Aim 1, we will determine the role for ATOH1 silencing in tumor initiation and its association with mutator CRC phenotypes. We will determine whether ATOH1 silencing in CRC requires mutation or methylation and define the molecular phenotype of ATOH1-silenced CRCs in primary human tumor samples provided by our collaborators at Vanderbilt University. We will directly test if Atoh1 mutation contributes to tumor initiation in transgenic mice, by quantifying early neoplastic changes in Atoh1- mutant colonic epithelia following carcinogen treatment. In Aim 2, we will test whether ATOH1 is required for the anti-cancer effect of Notch inhibitors (GSIs) in colon cancer. GSIs represent an exciting but mechanistically less defined approach to treat cancer. We predict that ATOH1 silencing will prevent GSI therapy from working in CRCs. We will test whether Atoh1 is required for the anti-proliferative effect of GSIs, both in mouse tumors and in cancer cell lines with multiple defined cancer phenotypes. We will also test a novel therapeutic approach to restore sensitivity to GSIs in ATOH1-silenced cells by re-expression of ATOH1. In Aim 3, we will define the mechanism of ATOH1's tumor suppressive function by testing the hypothesis that ATOH1 directs cell cycle exit via its target transcription factor SPDEF. Here, we will use complementary approaches in colon cancer cell lines and transgenic mice to determine whether SPDEF directs cell cycle arrest. Similarly, we will define the requirement for SPDEF induction to mediate ATOH1/GSI-induced growth arrest in knockout mice and CRC cells. CRCs constitute a major public health burden, with a need for novel therapies based on understanding of the molecular pathogenesis of cancer. The results of this project will: 1) identify a novel subtype-ATOH1- silenced CRC-which are likely refractory to GSI treatment; 2) define the mechanism of ATOH1 silencing and strategies to restore GSI sensitivity in ATOH1-silenced CRCs; 3) identify the mechanism of ATOH1 tumor suppression and downstream therapeutic targets (SPDEF). Thus, we will define the molecular mechanisms underpinning a new approach-GSI-induced growth arrest via ATOH1:SPDEF-to anti-cancer therapy.